Subcellular fractionation and proteomics of nuclear envelopes

Laurence Florens, Nadia Korfali, Eric C Schirmer

Research output: Contribution to journalArticlepeer-review

Abstract

Because of its many connections to other cell systems, the nuclear envelope (NE)is essentially impossible to purify to homogeneity. To circumvent these problems, we developed a subtractive proteomics approach in which the fraction of interest and a fraction known to contaminate the fraction of interest are separately analyzed, and proteins identified in both fractions are subtracted from the data set. This requires that the contaminating fraction can be purified to homogeneity. In this case, microsomal membranes (MMs) are used to represent endoplasmic reticulum contamination, allowing the identification of transmembrane proteins specific to the NE. To circumvent problems commonly associated with analyzing membrane proteins, the multidimensional protein identification technology (MudPIT) proteomics methodology is employed.
Original languageEnglish
Pages (from-to)117-37
Number of pages21
JournalMethods in Molecular Biology
Volume432
DOIs
Publication statusPublished - 2008

Keywords

  • Animals
  • Cell Fractionation
  • Chromatography, Liquid
  • Electrophoresis, Capillary
  • Intracellular Membranes
  • Liver
  • Mass Spectrometry
  • Mice
  • Microsomes, Liver
  • Nuclear Envelope
  • Proteins
  • Proteomics
  • Subcellular Fractions

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